Akiba S, Dodia C, Chen X, Fisher A B
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Comp Biochem Physiol B Biochem Mol Biol. 1998 Jun;120(2):393-404. doi: 10.1016/s0305-0491(98)10046-9.
An acidic Ca(2+)-independent phospholipase A2 (aiPLA2) has been isolated previously from rat lung and a human cDNA has been described. This study applied the method to larger scale isolation of the native protein from the bovine lung. A polyclonal antibody was generated to a 15 amino acid synthetic peptide based on a conserved rat/human sequence. This antibody recognized a single protein band with an estimated molecular mass of approximately 29 kDa in a soluble fraction obtained from bovine lung homogenate. A 29 kDa protein that reacted with the aiPLA2 antipeptide antibody was detected in fractions containing aiPLA2 activity on sequential column chromatographies. The partially purified enzyme showed 176-fold increase over the homogenate in Ca(2+)-independent PLA2 activity at pH 4. Activity was maximal with phosphatidylcholine substrate and was significantly less with phosphatidylethanolamine and anionic phospholipids. The enzyme had no acyl group preference in phosphatidylcholine and showed no preference for oxidized substrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiPLA2 activity was inhibited by a transition state phospholipid analog (MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine protease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacyl bromide, and ATP. Analysis of N-terminal amino acid sequence for the 29 kDa protein demonstrated its high homology to human 26 kDa aiPLA2. These was no significant change in molecular mass of the protein following treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA2 immunoreactivity with lamellar body, lysosomal, and cytosolic fractions. These results indicate isolation from bovine lung of a 29 kDa acidic Ca(2+)-independent phospholipase A2 homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholine.
一种酸性非钙依赖型磷脂酶A2（aiPLA2）先前已从大鼠肺中分离出来，且已有关于其人类cDNA的描述。本研究应用该方法从牛肺中大规模分离天然蛋白。基于大鼠/人类保守序列合成了一段15个氨基酸的肽段，并以此制备了多克隆抗体。该抗体在从牛肺匀浆获得的可溶部分中识别出一条估计分子量约为29 kDa的单一蛋白条带。在连续柱层析中，在含有aiPLA2活性的组分中检测到一种与aiPLA2抗肽抗体发生反应的29 kDa蛋白。部分纯化的酶在pH 4时，其非钙依赖型磷脂酶A2活性比匀浆增加了176倍。以磷脂酰胆碱为底物时活性最高，而以磷脂酰乙醇胺和阴离子磷脂为底物时活性明显较低。该酶对磷脂酰胆碱中的酰基没有偏好，对氧化底物也无偏好，但对1-O-烷基磷脂酰胆碱的活性较低。aiPLA2活性受到过渡态磷脂类似物（MJ33，1-十六烷基-3-三氟乙基甘油-sn-2-磷酸甲醇）、丝氨酸蛋白酶抑制剂和抗肽抗体的抑制，但对花生四烯酰三氟甲基酮、溴烯醇内酯、对溴苯甲酰溴和ATP不敏感。对该29 kDa蛋白的N端氨基酸序列分析表明，它与人类26 kDa的aiPLA2具有高度同源性。用内切糖苷酶F处理后，该蛋白的分子量没有显著变化。大鼠肺亚细胞组分的蛋白质印迹显示，aiPLA2与板层小体、溶酶体和胞质组分有免疫反应性。这些结果表明从牛肺中分离出了一种29 kDa的酸性非钙依赖型磷脂酶A2，它是大鼠和人类酶的同系物，并为肺表面活性物质磷脂酰胆碱代谢的特异性提供了证据。
